Alpert and Regnier in J. Chromatogr. 185, 375-392 (1979) have shown that polyethyleneimine (PEI) may be adsorbed to silica surfaces, thereby providing sufficient primary and secondary imino groups on adjacent adsorbed PEI molecules to be crosslinked by multifunctional oxiranes into a polymeric layer. Recently, the separation of synthetic oligonucleotides using high-performance liquid chromatography (HPLC) with columns of microparticulate silica coated with crosslinked polyethyleneimine has been reported in the literature by T. G. Lawson et al., Anal. Biochem. 133, 85-93 (1983).
More recently, new non-crosslinked covalently bound polyethyleneiminopropyl trimethoxy silane silica gel (PEI-PrSi-Silica gel) and polyethyleneiminopropyl trimethoxy silane controlled pore glass (PEI-PrSi-CPG) bonded phase products have been described by Hugh Ramsden in his U.S. Pat. No. 4,540,486, issued Sept. 10, 1985, as being useful in the separation and analysis of protein mixtures.
Since Ramsden's non-crosslinked covalently bound PEI-PrSi-Silica gel and PEI-PrSi-CPG products constitute the substrates to be N-acylated in accordance.with this invention, the disclosure of said U.S. Pat. No. 4,540,486 is incorporated herein in its entirety by reference thereto.
While said PEI-PrSi-Silica gel and PEI-PrSi-CPG bonded phase products have been found to be quite useful as solid phases for column packing in liquid chromatography for the separation and purification of proteins, these bonded phases products have been found not to be sufficiently strong cation exchangers for certain proteins and therefore separation and purification of such proteins has been rendered difficult or impossible. This has been found to be especially the case for separation and purification of proteins having low isoelectric points, that is, below about 5 or so. For example, said bonded phases do not provide sufficiently acceptable separation and purification of proteins such as ovalbumin and bovine serum albumin, for example.
It is, therefore, highly desirable that solid bonded phase products be provided that will permit suitable separation of proteins having isoelectric points of below about 5 and are strong cation exchangers for certain proteins. Additionally, it is highly desirable that there be provided such solid bonded phase products that are substantially fully charged at various pH's of from 2 and above and which provide a greater degree of selectivity than previously permissible. Moreover, it would be advantageous to provide solid bonded phase products that bind proteins more strongly than heretofore and which provide higher capacity by introducing SO.sub.3.sup.- groups adjacent to each COO.sup.- location. Furthermore, it would be highly advantageous that solid bonded phase products be provided that bind proteins with isoelectric points less than 5 due to the sulfonic acid group and also bind proteins with isoelectric points greater than 5 due to the combined binding strengths of both the sulfonic acid and carboxylic acid group.